We are interested in understanding the regulation of enhancer dependent gene expression in vivo. Specifically, there are two major areas of interest: (1) regulation of gene expression in undifferentiated cells and (2) interaction(s) of protein factors that may affect enhancer dependent expression. Our studies have focused on the role of DNA binding proteins in the regulation of gene transcription. In particular, we have examined the properties of nuclear factor I (NFI), a protein that has been shown by others to be important in both eukaryotic DNA replication and transcription. Preliminary results indicate: (1) NFI plays little or no role in activating expression of normally silent genes in undifferentiated cells. (2) The juxtaposition of NFI between enhancer and a down-stream promoter does not affect enhancer dependent transcription. (3) When NFI binding sites are placed at a distal 3' position, an increase in enhancer dependent transcription is seen. As an adjunct to elucidating the roles of DNA binding proteins in gene regulation, we are also studying the effects of prokaryotic DNA binding proteins and binding sequences placed in a eukaryotic environment.